The present invention relates to methods for isolating a defined quantity of a DNA target material from other substances in a medium to produce a suitable quantity of isolated DNA target material for further processing or analysis. The present invention particularly relates to methods for isolating a defined amount of DNA target material using a silica-containing solid support capable of reversibly binding a definable quantity of the DNA target material, such as magnetically responsive particles comprising silica or a silica derivative.
Many analysis techniques which involve the testing of a DNA target material present in a particular medium only work well when the DNA target material is isolated from other material in the medium, and quantified after isolation therefrom. Isolation of a DNA target material from other components in a forensics sample (e.g. bodily fluids collected from a crime scene, blood or buccal cells collected from suspects, etc.) is critical to ensure that the other components present in the sample do not interfere with analysis of the DNA target material. Unfortunately, forensic samples are frequently so small or so degraded that quantitation of DNA target material isolated therefrom can be time consuming and difficult. Moreover, the variance between individuals in the amount of leukocytes present in a given volume of blood further increases the variance of the quantity of DNA isolated.
With the advent of DNA typing as a tool for paternity testing and for identification of biological samples present at crime scenes has come the need to develop reliable methods for isolating and quantifying small amounts of genomic DNA. In the United States, the need to develop such systems has come from Federal Bureau of Investigation establishment of a database of analytical results from thirteen short tandem repeat (xe2x80x9cSTRxe2x80x9d) loci of human genomic DNA. These results are entered into a centralized database referred to as the Combined DNA Index System (xe2x80x9cCODISxe2x80x9d). STR analysis systems are based upon the use of amplification reactions, which enable one to analyze very small amounts of DNA, even sub-nanogram amounts. However, amplification only works well when the amount of DNA to be amplified is within a defined range, and when it is substantially isolated from contaminants which can inhibit or interfere with the amplification reaction. Thus, before STR loci can be amplified and analyzed, the target DNA must be purified and quantitated to reduce the risk of observing amplification artifacts. Quantitation is important in other applications as well, such as DNA sequencing.
Procedures currently used to isolate and quantify genomic DNA for use in genetic identity typing are time consuming, and too disjointed to be amenable to automation. For example, the following procedure is typically used to isolate and quantify genomic DNA for amplification and analysis of STR loci, such as the CODIS loci. First, blood or buccal swabs are obtained from individuals using a variety of devices and volumes. Second, these samples are processed to isolate DNA of variable purity and integrity. Third, the DNA is quantitated for downstream procedures so that the appropriate amount can be used to avoid artifacts. Fourth, the DNA is amplified using reactions that include primers specific for each of the STR loci to be analyzed. Finally, the amplification products are analyzed on a gel or capillary electrophoresis system for genotype identification. For a commercial system designed for use in co-amplifying and analyzing all thirteen CODIS loci, see GenePrint(copyright) PowerPlex(trademark) 1.1 and GenePrint(copyright) PowerPlex(trademark) 2.1 systems (Promega Corporation, Madison, Wis.).
White blood cells are the primary source of DNA in the blood. There is considerable variability in the white blood cell content of blood, due either to variability between individuals or variability of samples from a given individual based on the health of the individual at the time the sample was obtained. A similar variability exists in buccal swab samples, compounded by variability in the type of swab used, and storage conditions of the sample before sample processing.
Both inside and outside the context of amplification of genomic DNA for DNA typing analysis, discussed above, with amplification via the polymerase chain reaction (PCR), too little template results in low band intensity or no resultant band amplification. Excess DNA template frequently results in overamplification. Overamplification is recognized by an excessive number of artifact peaks and stutter bandsxe2x80x94defined as a minor peak directly below a major allele peak. There may also be a high incidence of background activity and xe2x80x9cpull-upxe2x80x9d, defined as the inability to separate the different color bands in a multiplex. Reamplification of a lesser quantity of DNA may be required if excessive artifacts are present. Stutter bands are particularly pronounced when excess DNA is present and capillary electrophoresis is used for the separation of PCR amplification products. Also, as with sequencing, the generation of full length amplification products can be inhibited when too much template DNA is present. In other words, in PCR amplification, excess template DNA can lead to the presence of partially amplified fragments and low amounts of completely amplified products.
More specifically, when PCR or other amplification methods are used in forensic applications to amplify DNA, when too much DNA is amplified in a single reaction, the sample is overamplified and the signal strength of the anticipated bands tends to fall outside the desired range of the detector. Traditionally, these difficulties are minimized by quantification of DNA after its purification, which requires additional steps, time and cost. In genetic identity testing, the presence of DNA in excess of that recommended for the analysis system employed often leads to uninterpretable results; this can waste very limited samples, particularly in the case of forensic analysis.
Another DNA application which requires accurate quantitation of the nucleic acid is sequencing. Sequencing of DNA is best done on samples of target DNA which have been isolated from other material present in a medium which can interfere with the sequencing reaction. It is also necessary to quantify samples of target DNA prior to initiation of a sequencing reaction. For example, in the area of DNA sequencing, the amount of DNA template in the sequencing reaction must be within a fairly narrow range. For example, when using plasmid DNA, 150-300 ng of DNA is recommended when using automated sequencing with BigDye(trademark) Chemistry (Perkin Elmer Biosystems). When using PCR products as sequencing templates with the same sequencing system, 40-80 ng of DNA is recommended. Too much template may result in short sequence read length, poor resolution or higher error rates. With too little template, the signal strength is too weak for optimal sequence reading.
Plasmid DNA is typically a source of DNA for sequencing reactions. There is considerable variability in plasmid DNA content within a population of bacterial cultures due to such factors as variability in plasmid copy number per cell, variability in growth media used, and concentration of cell mass.
There are a variety of methods currently used to quantitate a DNA target material in a sample. One such method is spectrophotometric determination. In this method, absorbance readings of a sample of unknown concentration are taken at the wavelength corresponding to the maximum absorbance of the DNA target material. For example, absorbance at 260 nanometers (nm) (xe2x80x9cA260xe2x80x9d) is used to determine the concentration of DNA in a solution, while absorbance at 280 nm (xe2x80x9cA280xe2x80x9d)is used to determine the concentration of protein in a solution. An absorbance reading at 260 nm of 1 corresponds to about 50 micrograms (50 xcexcg) per milliliter (xcexcg/ml) for double-stranded DNA, 40 xcexcg/ml for single-stranded DNA and RNA, and about 20 xcexcg/ml for single-stranded oligonucleotides. The ratio between the readings at 260 nm and 280 nm (xe2x80x9cA260/A280xe2x80x9d) provides an estimate of the degree to which a given target nucleic acid has been isolated from proteins and any other materials which absorb at 280 nm. Pure nucleic acid preparations have A260/A280 values of at least about 1.8. A limitation of the spectrophotometric method is that it is not sensitive enough to be used to detect and quantitate low amounts of nucleic acid. If a nucleic acid concentration in a sample is less than about 500 nanograms per milliliter (ng/ml), or if the sample is contaminated with other substances that either absorb or quench ultraviolet irradiation, inaccurate results are obtained.
Another method for quantitating DNA after it is isolated is the use of intercalating dyes such as ethidium bromide, SyberGreen (Molecular Dynamics, Sunnyvale, Calif.) or PicoGreen (Molecular Probes, Eugene, Oreg.). Dyes are often used when there is not enough DNA to accurately measure spectrophotometrically. The amount of fluorescence of ethidum bromide, when visualized with an ultraviolet (UV) light source, is proportional to the total mass of DNA. Therefore, a standard curve of known amounts of DNA and a known amount of a sample of unknown concentration may be run into an agarose gel and the gel subsequently stained with ethidium bromide and viewed with a UV light. This type of gel is called a yield gel. The quantity of DNA in the sample can be estimated by comparing the fluorescence of the sample with the fluorescence of the standards. Similarly, this method can be performed in solution with DNA intercalating dyes. DNA levels as low as about 25 pg/ml may be detected with PicoGreen. A limitation of the yield gel method or the use of dyes to quantitate DNA in solution is that it requires a visual, spectrophotometric or fluorometric approximation of the yield by comparison to another DNA sample. The variability in results obtained using this method is high and it is also prone to error resulting from contaminating components in the DNA sample.
At least two commercial kits are available for the quantitation of low amounts of human genomic DNA after isolation. These are the ACES(trademark) 2.0 Human DNA Quantitation Probe Plus System produced by Life Technologies, Inc. (Gaithersburg, Md.) and the Quantiblot(copyright) Human DNA Quantitation Kit produced by PE Applied Biosystems (Foster City, Calif.). These kits are typically used in laboratories performing genetic identity testing with human DNA. The Quantiblot(copyright) system is based on hybridization of a primate-specific biotinylated oligonucleotide probe to isolated DNA samples. The detection can be either colorimetric or chemiluminescent; either detection method is able to quantitate from 0.15 to 10 ng of human DNA. However, the test takes up to two hours. Furthermore, the chemiluminescent method requires X-ray film and processing capabilities, and can only be used for DNA from primates. The ACES(trademark) System is a similar system in that it requires binding of the DNA sample to a membrane and hybridization to a human-specific DNA probe and visualization by luminescence. This system is able to quantitate from 0.04 to 40 ng of human DNA. Both of these systems,have the same limitation as that of intercalating dyes; namely, they require a visual approximation of the yield by comparison to another DNA sample.
There is a need in the art for methods capable of removing a defined amount of DNA target material from a sample containing an excess of DNA target material. These defined DNA quantities can then be subsequently used in techniques in which having excess DNA present is detrimental to obtaining interpretable results. Such techniques include, but are not limited to PCR amplification, STR analysis, DNA sequencing and genetic identity testing.
Further, existing quantitation systems are not easily automatable and frequently are sensitive to contaminants remaining in the DNA preparation. Because of the large number of samples projected to be analyzed and databased, a high throughput process linking conventional STR-based steps is desirable without sacrificing low throughput needs. A system for isolating DNA from samples that quantitates the DNA in the process of purification would eliminate a process step and would be a significant advance in the art. A process less sensitive to artifacts than conventional quantitation techniques would also be desirable.
The present invention permits adsorption of a DNA target material from a medium to a solid phase under defined condition, and transfer of defined quantities of the biological material into a second solution. Target DNA transferred into the second solution according to the present method can be used as templates for sequencing or as templates for amplification reactions without a separate quantitation step. Because this technique eliminates the requirement of separately quantitating isolated target material before downstream processing or analysis, the method saves time and lends itself to automation.
The present system involves isolation of DNA target material from other material in a cell sample using a magnetic particle based separation. This approach allows for flexibility in processing as the magnetic separation can be employed in either a low throughput manual format or a high throughput robotic format.
Briefly, in one aspect, the present invention comprises a method of isolating a defined quantity of a DNA target material from other material in a medium by (a) providing a medium including the DNA target material; (b) providing a discrete quantity of silica magnetic particles capable of reversibly binding a definable quantity of the DNA target material; (c) forming a complex of the silica magnetic particles and the DNA target material by combining the silica magnetic particles and the medium; (d) removing the complex containing the DNA target material from the medium by application of an external magnetic field; and (optionally) (e) separating the DNA target material from the complex by eluting the DNA target material, whereby a defined quantity of the DNA target material is obtained. Preferably, the quantity of DNA target material provided in step (a) is in excess of the reversible binding capacity of the particles. Depending on the subsequent application and the quantity of silica magnetic particles provided, the elution step may be unnecessary.
The above method may also be carried out using silica-containing solid supports other than silica magnetic particles. When using other silica-containing solid supports, the complex containing the DNA target material may be removed from the medium by a variety of methods, such as centrifugation or filtration.
A preferred practice of the method of the present invention comprises the following steps. A sample of a certain type of medium containing a DNA target material is mixed with magnetic particles in the presence of a chaotropic salt, wherein, the magnetic particles have a known or definable capacity for adsorbing the DNA target material from the type of medium. When the sample type is cells, the cells are lysed to release the DNA target material into solution, where it forms a complex with the particles. After washing away other cell components, the DNA target material may be eluted in a discrete volume resulting in a solution of defined DNA target material concentration. The present method is suitable for use in isolating DNA target material from a wide variety of different sample types, including but not limited to, whole blood, white blood cells, sperm cells, buccal cells, or bacterial cells. In a preferred embodiment, the amount of DNA present in the sample is in excess of the binding capacity of the particles. Such samples can be presented in any one of a number of different forms, including but not limited to, liquid form, freeze-dried, dried onto material found at a crime scene, or mounted on a solid support (e.g., cheek cells on a swab or blood cells on a paper filter). Additional steps may be employed, if necessary, to remove the cells from a solid support. The purified DNA target material may be stored in the elution solution or left attached to the magnetic particles. Thus, multiple samples of the DNA target material can be obtained and used when needed.
In another aspect, the present invention is a method of isolating a defined quantity of target DNA material from other materials in a medium using a preferred form of silica magnetic particle, i.e., a siliceous oxide-coated magnetic particle, wherein the preferred particles are capable of reversibly binding a definable quantity of the target DNA material per milligram of particle. This aspect of the invention comprises the following steps. A mixture is formed comprising the medium including the target DNA material, the siliceous oxide-coated magnetic particles, and a chaotropic salt. The salt concentration is sufficient to cause the target DNA material to adhere to the particles. The mixture is incubated, or allowed to remain in mixture, until DNA is adhered to the siliceous oxide-coated magnetic particles in the mixture. The siliceous oxide-coated magnetic particles are then removed from the mixture using a magnetic force. A defined quantity of the target DNA material is eluted from the siliceous oxide-coated magnetic particles by contacting the particles with an elution solution.
In a further aspect, the present invention is a kit for isolating a defined quantity of a DNA target material from a medium containing the same. The kit includes a discrete quantity of siliceous oxide-coated magnetic particles suspended in an aqueous solution in a first container, wherein the particles have the capacity to reversibly bind a definable quantity of the DNA target material from a medium for specific sample type. Optionally, the kit may include other components needed to isolate a defined quantity of DNA target material from a medium containing the same according to the methods of the present invention. For example, the kit may also contain a chaotropic salt in a second container and a wash solution in a third container and instructions.
Yet another aspect of the invention is a method of determining a calibration model for quantitating a DNA target material in a sample type of interest, the method comprising: (a) providing a first medium, wherein the first medium includes a discrete quantity of sample type of interest; (b) providing a second medium, wherein the second medium includes a different discrete quantity of sample type of interest; (c) mixing a first discrete quantity of silica magnetic particles with the first medium, wherein the silica magnetic particles are capable of reversibly binding a defined quantity of the DNA target material, thereby forming a first complex of the silica magnetic particles and the DNA target material from the first medium; (d) mixing a second discrete quantity of silica magnetic particles with the second medium, wherein the silica magnetic particles are capable of reversibly binding a defined quantity of the DNA target material, thereby forming a second complex of the silica magnetic particles and the DNA target material from the second medium; (e) removing the first complex from the first medium and the second complex from the second medium by application of an external magnetic field; (f) separately eluting the DNA target material from the first complex and second complex, producing a first eluent of isolated DNA target material from the first complex and a second eluent of isolated DNA target material from the second complex; (g) determining the amount of DNA target material in the first eluent and in the second eluent. Preferably, the first discrete quantity of particles provided in step (c) is the same quantity of particles as the second discrete quantity provided in step (d).
One calibration method, as illustrated in Example 3, involves determining the amount of particles necessary in the purification of target DNA from the smallest sample size (the smallest amount of DNA available) so that the DNA target material is present in excess, and the resulting purified target DNA is also in the desired target range. After determining the amount of particles desired from the smallest sample size, it is important to ensure that purification from the larger sample sizes also produces purified target DNA that is in the desired range of concentration or yield. This method generally determines the largest quantity of DNA that can be reliably obtained from the desired range of sample sizes, wherein the amount of target DNA obtained from each of the samples lies within the desired quantitative range of target DNA.
Another calibration method, as illustrated in Example 8, relies on using sample sizes that are known to contain a large excess of DNA target material, so that the range of particles used in the purification is known to be the factor limiting the quantity of DNA target material that is obtained. Using this method, a correlation is made between the highest and lowest quantity of target DNA that provides the desired utility for the application (in Example 8, this application is DNA sequencing), and the amount of particles used in the purification that results in the purification of target DNA quantities within the range desired for the application. When the target material is DNA, the amount of target material present in each eluent produced to construct the calibration model, as described above, is preferably determined by DNAQuant or PicoGreen analysis.
There are a variety of applications where this invention has utility. Two such areas include DNA sequencing, particularly automated DNA sequencing, and genetic analysis involving nucleic acid amplification reactions, such as the polymerase chain reaction (PCR). In each of these applications, the quantity of DNA target material must be kept within a well-defined range. Genetic analyses may include, for example, genetic identification used in forensics or paternity cases, and genetic analyses used in clinical laboratories. In such cases, it is helpful to have approximately the same quantity of DNA target material in each amplification reaction. Consistent quantity leads to consistent band intensity in a gel analysis and limited artifacts. The invention may also be used in conjunction with other amplification systems, such as transcription mediated amplification.
The present methods are readily adaptable to automation as, in a preferred practice, they allow for the simultaneous isolation and quantitation of DNA target material from multiple samples. For example, the present method could be used to isolate a defined quantity of target genomic DNA from blood or other tissue samples taken from multiple individuals in a population. Loci of interest, such as STR loci, of the isolated and quatitated genomic DNA could then be amplified and analyzed using any one of a number of known genetic analysis methods. See, for example, the GenePrints(copyright) STR analysis systems from Promega Corporation. When used as described immediately above to isolate, quantitate, and co-amplify multiple STR loci, such as the CODIS loci, in multiplex reactions (e.g., using the GenePrint(copyright) PowerPlex System from Promega) the amount of information in databases of such DNA typing results could be rapidly increased. The more data present in such databases, the more useful the databases are for identification of individuals, particularly for forensics applications.
The DNA target material isolated using the method of the present invention is sufficiently free of contaminating material for additional processing or analysis using standard molecular biology techniques. Applications of the present methods to isolate and quantitate various different DNA target materials from a variety of different media will become apparent from the detailed description of the invention below.
Other features, advantages and applications of the invention will become apparent to those skilled in the art upon review of the following detailed description and claims.